Age-related macular degeneration (AMD) is the major cause of blindness in those over 60 years. In AMD, retinal pigment epithelial (RPE) cells become dysfunctional and degenerate, which leads to the loss of photoreceptors and, ultimately, central vision. Several studies have correlated AMD susceptibility with low pigment level in RPE cells. The production of pigment, or melanogenesis, is completed by birth in RPE, and the pigment melanin degrades with aging, further implicating the contribution of low pigment level to the development of AMD later in life. How pigmentation might protect individuals from developing AMD is an important unanswered question. Studies addressing the mechanism and functional consequences of pigment loss have been hampered by lack of appropriate human model systems, yet these are important issues to address because the ability to reactivate melanogenesis could be protective not only by reducing sensitivity to light-induced toxicity but also by preserving key functions of the RPE cells like the phagocytosis of shed photoreceptor outer segments. These questions will be addressed in this proposal by utilizing an approach made possible through the development of human induced pluripotent stem cell (iPSC) technologies. We will reprogram aged human RPE cells to human induced pluripotent stem cells (hiPSCs). The reprogramming process can reset the molecular clock of the resulting hiPSCs and their derivatives to an immature, young-like state. Thus, this system provides the unique opportunity to investigate the mechanisms underlying reactivation of melanogenesis following the rejuvenation of aged RPE cells to young-like hiPSC-derived RPE (hiPSC-RPE) cells. Preliminary data suggests that pigment level increases in hiPSC-RPE cells compared to aged RPE cells from the same donor. We will characterize melanogenesis in several isogenic aged RPE and hiPSC-RPE cell pairs. Gene expression analysis will be used to examine the melanogenesis pathway to determine whether there is a developmental switch that regulates silencing. Taking advantage of their young-like status following reprogramming, hiPSC-RPE cells will be matured in vitro to observe when pigment synthesis ceases and to identify molecular strategies that could reactivate melanogenesis. Finally, hiPSC-RPE cells will be genetically engineered using CRISPR/Cas9 technology to reduce biogenesis of melanosomes, the melanin-containing organelles, through loss of GPR143, or to reduce melanin synthesis through loss of TYR; these engineered lines will serve to mimic the loss of pigment with aging. The genetically engineered loss-of-function hiPSC-RPE cells will be compared to untargeted isogenic control hiPSC-RPE cells for their vulnerability to light-induced toxicity and their abilit to phagocytose photoreceptor outer segments. This will determine for the first time in a human model system whether affecting melanosome number or melanin content alters these key RPE functions. The goal of this proposal is to understand the developmental switch that regulates melanogenesis and to lay the groundwork for determining how melanogenesis might be stimulated to prevent or treat AMD.